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Purity: ≥98%
Afatinib (formerly BIBW 2992; BIBW-2992; brand name: Gilotrif), is a potent, covalent/irreversible, and orally bioavailable dual (EGFR/ErbB) receptor tyrosine kinase (RTK) inhibitor with anticancer activity. Afatinib is an FDA-approved anticancer medication used to treat lung cancer that is not small cell (NSCLC). In the USA, Gilotrif is the brand name under which it is sold. It is 100 times more active against the Gefitinib-resistant L858R-T790M EGFR mutant. It irreversibly binds to and inhibits EGFR/HER2, including EGFR(wt), EGFR(L858R), EGFR(L858R/T790M), and HER2. In cell-free assays, its IC50 values are 0.5 nM, 0.4 nM, 10 nM, and 14 nM, respectively.
| Targets |
EGFRL858R (IC50 = 0.4 nM); EGFR (wt) (IC50 = 0.5 nM); ErbB4 (IC50 = 1 nM); EGFRL858R/T790M (IC50 = 10 nM); HER2 (IC50 = 14 nM)
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| ln Vitro |
BIBW2992 exhibits potent inhibition of EGFR and HER2 in both wild-type and mutant forms. It has comparable potency to gefitinib against L858R EGFR, but it is approximately 100 times more active against the L858R-T790M EGFR double mutant that is resistant to gefitinib. In vivo, BIBW2992 demonstrates strong effects on the phosphorylation of both EGFR and HER2. When compared to reference compounds (like Lapatinib et al.), it performs well in all tested cell types, including human epidermoid carcinoma cell line A431 that expresses EGFR, murine NIH-3T3 cells transfected with HER2, breast cancer cell line BT-474, and gastric cancer cell line NCI-N87 that express endogenous HER2.[1]
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| ln Vivo |
Afatinib (0-20 mg/kg, Orally, daily for 25 days) exhibits a significant decrease in tumor growth and phosphorylation of AKT, HER2, EGFR, and HER3.
Afatinib (15 mg/kg, Orally, in a schedule of 5 days on plus 2 days off, for two weeks) strongly inhibits the growth of HKESC-2 tumor. |
| Enzyme Assay |
The human EGFR wild type and EGFR L858R/T790M double mutant tyrosine kinase domains are fused to GST and extracted. Next, enzyme activity is measured with and without the inhibitor BIBW2992, which is serially diluted in 50% DMSO. Biotinylated pEY (bio-pEY) is added as a tracer substrate and a random polymer, pEY (4:1), is used as the substrate. Utilizing the baculovirus system, the HER2 kinase domain is cloned and extracted in a manner akin to that of EGFR kinase. Supplementary information contains specifics about the assays conducted for EGFR, HER2, SRC, BIRK, and VEGFR2 kinase activity.
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| Cell Assay |
For the EGFR phosphorylation test, 1 × 104 NSCLC cells are plated into each well of a 96-well plate and grown for an entire night in serum-free medium. The following day, the plates are incubated at 37 °C for one hour following the addition of BIBW2992. EGF stimulation is carried out at room temperature for 10 minutes using 100 ng/mL. Following an hour of shaking at room temperature and an extraction using 120 μL of HEPEX buffer per well, the cells are cleaned with ice-cold PBS. HER2 phosphorylation assay uses 2 × 104 cells per well in total. The c-erb2/HER2 oncoprotein Ab-5(Clone N24)-Biotin and anti-EGFR-Biotin are coated on streptavidin precoated plates at a 1:100 dilution in blocking buffer. Once in the antibody-coated wells, cell extracts are allowed to sit at room temperature for one hour. Measurement of extinction occurs at 450 nm.
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| Animal Protocol |
Athymic NMRI-nu/nu female mice[1]
20 mg/kg Oral administration Four bitransgenic mice on continuous doxycycline diets for more than 6 weeks were subjected to MRI (Figure 4) to document the lung tumor burden. Afatinib (BIBW2992) formulated in 0.5% methocellulose-0.4% polysorbate-80 (Tween 80) was administered orally by gavage at 20 mg/kg once daily dosing schedule. Rapamycin was dissolved in 100% ethanol, freshly diluted in 5% PEG400 and 5% Tween 80 before treatment and administered by intraperitoneal injection at 2 mg/kg daily dosage. Mice were monitored by MRI every 1 or 2 weeks to determine reduction in tumor volume and killed for further histological and biochemical studies after drug treatment. For immunohistochemistry staining, three tumor-bearing mice in each group were treated three times with either Afatinib (BIBW2992) (20 mg/kg) alone or Afatinib (BIBW2992) (20 mg/kg) and rapamycin 2 mg/kg at 24 h intervals and killed 1 h after the last drug delivery. All the mice were kept on the doxycycline diet throughout the experiments. Littermates were used as controls.[1] |
| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion
Following oral administration, time to peak plasma concentration (Tmax) is 2 to 5 hours. Maximum concentration (Cmax) and area under the concentration-time curve from time zero to infinity (AUC0-∞) values increased slightly more than dose proportional in the range of 20 to 50 mg. The geometric mean relative bioavailability of 20 mg tablets was 92% as compared to an oral solution. Additionally, systemic exposure to afatinib is decreased by 50% (Cmax) and 39% (AUC0-∞), when administered with a high-fat meal compared to administration in the fasted state. Based on population pharmacokinetic data derived from clinical trials in various tumor types, an average decrease of 26% in AUCss was observed when food was consumed within 3 hours before or 1 hour after taking afatinib. In humans, excretion of afatinib is primarily via the feces. Following administration of an oral solution of 15 mg afatinib, 85.4% of the dose was recovered in the feces and 4.3% in urine. The parent compound afatinib accounted for 88% of the recovered dose. The volume of distribution of afatinib recorded in healthy male volunteers is documented as 4500 L. Such a high volume of distribution in plasma suggests a potentially high tissue distribution. The apparent total body clearance of afatinib as recorded in healthy male volunteers is documented as being a high geometric mean of 1530 mL/min. Metabolism / Metabolites Enzyme-catalyzed metabolic reactions play a negligible role for afatinib in vivo. Covalent adducts to proteins were the major circulating metabolites of afatinib. Biological Half-Life Afatinib is eliminated with an effective half-life of approximately 37 hours. Thus, steady-state plasma concentrations of afatinib were achieved within 8 days of multiple dosing of afatinib resulting in an accumulation of 2.77-fold (AUC0-∞) and 2.11-fold (Cmax). In patients treated with afatinib for more than 6 months, a terminal half-life of 344 h was estimated. |
| Toxicity/Toxicokinetics |
Hepatotoxicity
Elevations in serum aminotransferase levels are common during afatinib therapy occurring in 20% to 50% of patients, but rising above 5 times the upper limit of the normal range in only 1% to 2%. Hepatic failure is said to have occurred in 0.2% of patients and to have resulted in several fatalities. Hepatotoxicity appears to be a class effect among protein kinase inhibitors of EGFR2, although liver injury appears to be more frequent and more severe with gefitinib than with afatinib and erlotinib. Specific details of the liver injury associated with afatinib such as latency, serum enzyme pattern, clinical features and course, have not been published. Other EGFR inhibitors, such as erlotinib and gefitinib typically cause liver injury arising within days or weeks of starting therapy and presenting abruptly with hepatocellular enzyme elevations and a moderate-to-severe course. Immunoallergic and autoimmune features are not common. The rate of clinically significant liver injury and hepatic failure is increased in patients with preexisting cirrhosis or hepatic impairment due to liver tumor burden. Likelihood score: D (possible cause of clinically apparent liver injury). Effects During Pregnancy and Lactation ◉ Summary of Use during Lactation No information is available on the clinical use of afatinib during breastfeeding. Because afatinib is about 95% bound to plasma proteins, the amount in milk is likely to be low. However, its half-life is about 37 hours and it might accumulate in the infant. the manufacturer recommends that breastfeeding be discontinued during afatinib therapy and for 2 weeks after the last dose. ◉ Effects in Breastfed Infants Relevant published information was not found as of the revision date. ◉ Effects on Lactation and Breastmilk Relevant published information was not found as of the revision date. Protein Binding In vitro binding of afatinib to human plasma proteins is approximately 95%. Afatinib binds to proteins both non-covalently (traditional protein binding) and covalently. |
| References | |
| Additional Infomation |
Pharmacodynamics
Aberrant ErbB signaling triggered by receptor mutations, and/or amplification, and/or receptor ligand overexpression contributes to the malignant phenotype. Mutation in EGFR defines a distinct molecular subtype of lung cancer. In non-clinical disease models with ErbB pathway deregulation, afatinib as a single agent effectively blocks ErbB receptor signaling resulting in tumor growth inhibition or tumor regression. NSCLC tumors with common activating EGFR mutations (Del 19, L858R) and several less common EGFR mutations in exon 18 (G719X) and exon 21 (L861Q) are particularly sensitive to afatinib treatment in non-clinical and clinical settings. Limited non-clinical and/or clinical activity was observed in NSCLC tumors with insertion mutations in exon 20. The acquisition of a secondary T790M mutation is a major mechanism of acquired resistance to afatinib and gene dosage of the T790M-containing allele correlates with the degree of resistance in vitro. The T790M mutation is found in approximately 50% of patients' tumors upon disease progression on afatinib, for which T790M targeted EGFR TKIs may be considered as a next line treatment option. Other potential mechanisms of resistance to afatinib have been suggested preclinically and MET gene amplification has been observed clinically. At the same time, the effect of multiple doses of afatinib (50 mg once daily) on cardiac electrophysiology and the QTc interval was evaluated in an open-label, single-arm study in patients with relapsed or refractory solid tumors. Ultimately, no large changes in the mean QTc interval (i.e., >20 ms) were detected in the study. |
| Molecular Formula |
C24H25CLFN5O3
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|---|---|
| Molecular Weight |
485.94
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| Exact Mass |
485.162
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| Elemental Analysis |
C, 59.32; H, 5.19; Cl, 7.30; F, 3.91; N, 14.41; O, 9.88
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| CAS # |
850140-72-6
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| Related CAS # |
Afatinib dimaleate;850140-73-7;Afatinib-d6;1313874-96-2;Afatinib oxalate;1398312-64-5;(R)-Afatinib;439081-17-1;Afatinib-d4
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| PubChem CID |
10184653
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| Appearance |
White to light yellow solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
676.9±55.0 °C at 760 mmHg
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| Melting Point |
100 - 102 °C
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| Flash Point |
363.2±31.5 °C
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| Vapour Pressure |
0.0±2.1 mmHg at 25°C
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| Index of Refraction |
1.668
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| LogP |
3.59
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
8
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| Heavy Atom Count |
34
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| Complexity |
702
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| Defined Atom Stereocenter Count |
1
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| SMILES |
N(C1C=CC(F)=C(Cl)C=1)C1=NC=NC2=CC(=C(C=C12)NC(=O)/C=C/CN(C)C)O[C@@H]1COCC1
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| InChi Key |
ULXXDDBFHOBEHA-CWDCEQMOSA-N
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| InChi Code |
InChI=1S/C24H25ClFN5O3/c1-31(2)8-3-4-23(32)30-21-11-17-20(12-22(21)34-16-7-9-33-13-16)27-14-28-24(17)29-15-5-6-19(26)18(25)10-15/h3-6,10-12,14,16H,7-9,13H2,1-2H3,(H,30,32)(H,27,28,29)/b4-3+/t16-/m0/s1
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| Chemical Name |
(E)-N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)-oxolan-3-yl]oxyquinazolin-6-yl]-4-(dimethylamino)but-2-enamide
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| Synonyms |
BIBW2992; Afatinib free base; BIBW 2992; BIBW 2992; Afatinib; trade name: Gilotrif, Tomtovok and Tovok
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.14 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.14 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.14 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Solubility in Formulation 4: 2% DMSO+30% PEG 300+5% Tween 80+ddH2O: 10 mg/mL Solubility in Formulation 5: 5 mg/mL (10.29 mM) in 0.5% Methylcellulose/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.0579 mL | 10.2893 mL | 20.5787 mL | |
| 5 mM | 0.4116 mL | 2.0579 mL | 4.1157 mL | |
| 10 mM | 0.2058 mL | 1.0289 mL | 2.0579 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT05154396 | Active Recruiting |
Drug: Afatinib | Chordoma | Leiden University Medical Center | June 21, 2018 | Phase 2 |
| NCT03827070 | Active Recruiting |
Drug: Afatinib Drug: Talcum powder |
Non Small Cell Lung Cancer | Center Trials & Treatment Europe | March 5, 2019 | Phase 1 |
| NCT04413201 | Active Recruiting |
Drug: Afatinib Drug: Osimertinib |
Non-squamous NSCLC | Michael Hopp | September 11, 2020 | Phase 4 |
| NCT05267288 | Recruiting | Drug: afatinib | PFS | Qingdao Central Hospital | February 1, 2021 | Phase 2 |
| NCT05519917 | Not yet recruiting | Drug: Afatinib | Chordoma of Clivus | Huashan Hospital | October 1, 2022 | Phase 2 |
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Afatinib covalently binds to cysteine number 797 of the epidermal growth factor receptor (EGFR) via a Michael addition (IC50 = 0.5 nM).Schubert-Zsilavecz, M, Wurglics, M,Neue Arzneimittel Frühjahr 2013.(in German) |
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